Infectious diseases caused by pathogenic agents, such as bacteria, viruses (e.g., viral hemorrhagic fevers (VHFs)), and parasites pose a significant public health problem, due to the severity of the diseases, high lethality, inter-human contagiousness of certain agents, and lack of effective treatment for most of them.
Control of epidemics crucially depends on the rapid detection and accurate identification of the agent, in order to define and implement timely and appropriate action. In this context, it is essential to produce and validate tools for early detection of outbreaks, precise identification of the etiologic agent, and improved disease surveillance.
In this respect, detection of antibodies in body fluids constitutes a major part of the diagnosis of virally induced diseases, other diseases caused by infectious organisms, autoimmune diseases and the detection of cancer. As a matter of fact, certain antibodies can serve as markers in diagnosis and can lead to prognosis and treatment, as their presence are known to correlate with the outbreak of a pathogen. This is particularly the case for the antibodies targeting viral antigens exclusively.
Current methods for detecting the presence of antibodies include diverse techniques such as immunofluorescence microscopy, chemiluminescence assay, Western blotting, Radio Immuno-Precipitation assay (RIP) and ELISA. The parallel detection of several antibodies simultaneously may be particularly useful by minimizing the matrix effects that exist between individual assays, such as in ELISAs, because the calibrators and the antibodies are analyzed under the same conditions; it therefore will generate comparable results for the measurement of multiple antibodies present within the same sample. One such assay is the Luminex multiplex assay utilizing antigens bound to beads. Pickering et al., Clinical and Diagnostic Laboratory Immunology 9:872-876 (2002).
Complicating the straightforward identification of pathogenically relevant antibodies, however, is that normal sera contain large amounts of natural antibodies which manifest themselves in complex staining patterns (Avrameas S. Immunol. Today 1991). The presence of these natural antibodies can complicate the differentiation of disease-associated antibodies from the complex background of “auto-immune noise”, i.e. naturally occurring autoantibodies. As noted in Anderson et al., Methods Mol. Biol. 723:227-238 (2011), cross-talk and interference remain a concern with multiplex assays.
Binding of human antibodies directly to the beads in a multiplex assay has also been reported as a problem. Tainsky et al., Biomarker Insights 2:261-267 (2007); Waterboer et al., J. Immunol. Methods 309:200-204 (2006).
Moreover, a low difference between positive and control samples can limit the utility of multiplex assays. Burbelo et al., Exp. Rev Vaccines, 9:567-578 (2010).
Another complication is that ELISA and multiplex assays do not necessary give the same results. For example, Pickering et al., 2002, noted numerous discrepancies between the two assays when measuring protective antibodies against various viral and bacterial antigens. This may reflect alterations in antigenicity relating to how the antigen is attached to the substrate. Ambrosino et al., Malaria Journal 9:317 (2010). Similarly, Cham et al., Malaria Journal 7:108 (2008), reported that some samples showed a marked difference between the ELISA and bead-based assay readings, some of which had higher ELISA readings. This could be due to the fact that different parts of the recombinant proteins are accessible by antibodies when the proteins are bound to a surface or a sphere. Id.
The preparation of sufficient quantities of the antigen for commercial applications (i.e., kits) is also required. Burbelo et al., Exp. Rev Vaccines, 9:567-578 (2010).
In view of the foregoing, there exists a need for addressable systems and methods, which can provide additional improvements in high throughput, cost-effectiveness, and accuracy for molecular diagnosis of antibody-generating diseases. The present invention satisfies these and other needs.